Method of determining dihydropyrimidine dehydrogenase gene expression

ABSTRACT

The present invention relates to prognostic methods which are useful in medicine, particularly cancer chemotherapy. The object of the invention to provide a method for assessing Dihydropyrimidine dehydrogenase (DPD) expression levels in tissues and prognosticate the probable resistance of a patient&#39;s tumor to treatment with 5-FU based therapies by examination of the amount of DPD mRNA in a patient&#39;s tumor cells and comparing it to a predetermined threshold expression level. More specifically, the invention provides to oligonucleotide primer pairs DPD3A and DPD3B and methods comprising their use for detecting levels of Dihydropyrimidine dehydrogenase (DPD) mRNA.

FIELD OF THE INVENTION

[0001] The present invention relates to prognostic methods which areuseful in medicine, particularly cancer chemotherapy. The invention alsorelates to assessment of gene expression of tumor cells of a patient.More specifically, the invention relates to oligonucleotides and methodscomprising their use for detecting levels of Dihydropyrimidinedehydrogenase (DPD) mRNA expression using RT-PCR.

BACKGROUND OF THE INVENTION

[0002] Cancer arises when a normal cell undergoes neoplastictransformation and becomes a malignant cell. Transformed (malignant)cells escape normal physiologic controls specifying cell phenotype andrestraining cell proliferation. Transformed cells in an individual'sbody thus proliferate in the absence of these normal controls, thusforming a tumor. When a tumor is found, the clinical objective is todestroy malignant cells selectively while mitigating any harm caused tonormal cells in the individual undergoing treatment.

[0003] Chemotherapy is based on the use of drugs that are selectivelytoxic (cytotoxic) to cancer cells. Several general classes ofchemotherapeutic drugs have been developed, including drugs thatinterfere with nucleic acid synthesis, protein synthesis, and othervital metabolic processes.

[0004] 5-Fluorouracil (5-FU) is a very widely used drug used for thetreatment of many different types of cancers, including major cancerssuch as those of the GI tract and breast (Moertel, C. G. New Engl. J.Med., 330:1136-1142, 1994). 5-FU as a single agent was for more than 40years the standard first-line treatment for colorectal cancer, but itwas supplanted as “standard of care” by the combination of 5-FU andCPT-11 (Saltz et al., Irinotecan Study Group. New England Journal ofMedicine. 343:905-14, 2000). Recently, the combination of 5-FU andoxaliplatin has produced high response rates in colorectal cancers(Raymond et al., Semin. Oncol., 25:4-12, 1998). Thus, it is likely that5-FU will be used in cancer treatment for many years because it remainsthe central component of current chemotherapeutic regimens. In addition,single agent 5-FU therapy continues to be used for patients in whomcombination therapy with CPT-11 or oxaliplatin is likely to beexcessively toxic.

[0005] 5-FU is typical of most anti-cancer drugs in that only theminority of patients experience a favorable response to the therapy.Large randomized clinical trials have shown the overall response ratesof tumors to 5-FU as a single agent for patients with metastaticcolorectal cancer to be in the 15-20% range (Moertel, C. G. New Engl. J.Med., 330:1136-1142, 1994). In combination with other chemotherapeuticsmentioned above, tumor response rates to 5-FU-based regimens have beenincreased to almost 40%. Nevertheless, the majority of treated patientsderive no tangible benefit from having received 5-FU based chemotherapy,and are subjected to significant risk, discomfort, and expense. Sincethere has been no reliable means of anticipating the responsiveness ofan individual's tumor prior to treatment, the standard clinical practicehas been to subject all patients to 5-FU-based treatments, fullyrecognizing that the majority will suffer an unsatisfactory outcome.

[0006] The mechanism of action and the metabolic pathway of 5-FU havebeen intensively studied over the years to identify the most importantbiochemical determinants of the drug's anti-tumor activity. The ultimategoal was to improve the clinical efficacy of 5-FU by a) modulation ofits intracellular metabolism and biochemistry and b) by measuringresponse determinants in patients' tumors prior to therapy to predictwhich patients are most likely to respond (or not to respond) to thedrug. Two major determinants emerged from these studies: 1) the targetenzyme of 5-FU, thymidylate synthase (TS) and 2) the catabolic enzymedihydropyrimidine dehydrogenase (DPD).

[0007] The first studies in the area of tumor response prediction to5-FU based therapy centered on the target enzyme TS in colorectalcancer. Leichman et al (Leichman et al., J. Clin Oncol., 15:3223-3229,1997) carried out a prospective clinical trial to correlate tumorresponse to 5-FU with TS gene expression as determined by RT-PCR inpre-treatment biopsies from colorectal cancers. This study showed: 1) alarge 50-fold range of TS gene expression levels among these tumors, and2) strikingly different levels of TS gene expression between respondingand non-responding tumors. The range of TS levels of the respondinggroups (0.5-4.1, relative to an internal control) was narrower than thatof the non-responding groups (1.6-23.0, relative to an internalcontrol). The investigators determined a resulting “non-response cutoff”threshold level of TS expression above which there were onlynon-responders. Thus, patients with TS expression above this“non-response cutoff” threshold could be positively identified asnon-responders prior to therapy. The “no response” classificationincluded all therapeutic responses with <50% tumor shrinkage,progressing growth resulting in a >25% tumor increase andnon-progressing tumors with either <50% shrinkage, no change or <25%increase. These tumors had the highest TS levels. Thus, high TSexpression identifies especially resistant tumors. TS expression levelsabove a certain threshold identified a subset of tumors not respondingto 5-FU, whereas TS expression levels below this number predicted anappreciably higher response rate yet did not specifically identifyresponding tumors.

[0008] Subsequent studies investigated the usefulness of DPD expressionlevels as a tumor response determinant to 5-FU treatment in conjunctionwith TS expression levels. DPD is a catabolic enzyme which reduces the5,6 double bond of 5-FU, rendering it inactive as a cytotoxic agent.Previous studies have shown that DPD levels in normal tissues couldinfluence the bio-availability of 5-FU, thereby modulating itspharmacokinetics and anti-tumor activity (Harris et al., Cancer Res.,50: 197-201, 1990). Additionally, evidence has been presented that DPDlevels in tumors are associated with sensitivity to 5-FU (Etienne etal., J. Clin. Oncol., 13: 1663-1670, 1995; Beck et al., Eur. J. Cancer,30: 1517-1522, 1994). Salonga et al, (Clin Cancer Res., 6:1322-1327,2000) investigated gene expression of DPD as a tumor responsedeterminant for 5-FU/leucovorin treatment in a set of tumors in which TSexpression had already been determined. As with TS, the range of DPDexpression among the responding tumors was relatively narrow (0.6-2.5,4.2-fold; relative to an internal control) compared with that of thenon-responding tumors (0.2-16, 80-fold; relative to an internalcontrol). There were no responding tumors with a DPD expression greaterthan a threshold level of about 2.5. Furthermore, DPD and TS expressionlevels showed no correlation with one another, indicating that they areindependently regulated genes. Among the group of tumors having both TSand DPD expression levels below their respective “non-response cut-off”threshold levels, 92% responded to 5-FU/LV. Thus, responding tumorscould be identified on the basis of low expression levels of DPD and TS.

[0009] DPD is also an important marker for 5-FU toxicity. It wasobserved that patients with very low DPD levels (such as in DPDDeficiency Syndrome; i.e. thymine uraciluria) undergoing 5-FU basedtherapy suffered from life-threatening toxicity (Lyss et al., CancerInvest., 11: 2390240, 1993). Indeed, the importance of DPD levels in5-FU therapy was dramatically illustrated by the occurrence of 19 deathsin Japan from an unfavorable drug interaction between 5-FU and ananti-viral compound Sorivudine (Diasio et al., Br. J. Clin. Pharmacol.46, 1-4, 1998) It was subsequently discovered that a metabolite ofSorivudine is a potent inhibitor of DPD. This treatment resulted in DPDDeficiency Syndrome-like depressed levels of DPD which increased thetoxicity of 5-FU to the patients (Diasio et al., Br. J. Clin. Pharmacol.46, 1-4, 1998).

[0010] Thus, because of a) the widespread use of 5-FU protocols incancer treatment, b) the important role of DPD expression in predictingtumor response to 5-FU and c) the sensitivity of individuals withDPD-Deficiency Syndrome to common 5-FU based treatments, it is clearthat accurate determination of DPD expression levels prior tochemotherapy will provide an important benefit to cancer patients.

[0011] Measuring DPD enzyme activity requires a significant amount offresh tissue that contains active enzyme. Unfortunately, mostpre-treatment tumor biopsies are available only as fixed paraffinembedded (FPE) tissues, particularly formalin-fixed parafin embeddedtissues which do not contain active enzyme. Moreover, biopsies generallycontain only a very small amount of heterogeneous tissue.

[0012] RT-PCR primer and probe sequences are available to analyze DPDexpression in frozen tissue or fresh tissue. However, those primers areunsuitable for the quantification of DPD mRNA from fixed tissue byRT-PCR. Heretofore, existing primers give no or erratic results. This isthought to be due to the a) inherently low levels of DPD RNA; b) verysmall amount of tissue embedded in the paraffin; and c) degradation ofRNA in the paraffin into short pieces of <100 bp. As a result, otherinvestigators have made a concerted yet unsuccessful efforts to obtainoligonucleotide primer sets allowing for such a quantification of DPDexpression in paraffinized tissue. Thus, there is a need for method ofquantifying DPD mRNA from fixed tissue in order to provide an earlyprognosis for proposed cancer therapies. Because it has been shown thatDPD enzyme activity and corresponding mRNA expression levels are wellcorrelated (Ishikawa et al., Clin. Cancer Res., 5:883-889, 1999; Johnsonet al., Analyt. Biochem. 278: 175-184, 2000), measuring DPD mRNAexpression in FPE specimens provides a way to assess the DPD expressionlevels status of patients without having to determine enzyme activity infresh tissues. Furthermore, FPE specimens are readily amenable tomicrodissection, so that DPD gene expression can be determined in tumortissue uncontaminated with stromal tissue.

[0013] Accordingly, it is the object of the invention to provide amethod for assessing DPD levels in tissues and prognosticate theprobable resistance of a patient's tumor to treatment with 5-FU basedtherapies, by examination of the amount of DPD mRNA in a patient's tumorcells and comparing it to a predetermined threshold expression level.

SUMMARY OF THE INVENTION

[0014] In one aspect of the invention there is provided oligonucleotideprimers having the sequence of DPD3A-51F (SEQ ID NO: 1) or DPD3A-134R(SEQ ID NO:2), as well as oligionucleotide primers DPD3b-651F (SEQ IDNO: 7) and DPD3b-736R (SEQ ID NO: 8) and sequences substantiallyidentical thereto. The invention also provides for oligonucleotideprimers having a sequence that hybridizes with DPD3A-51F (SEQ ID NO: 1),DPD3A-134R (SEQ ID NO:2), DPD3b-651F (SEQ ID NO:7), DPD3b-736R (SEQ IDNO: 8) or complements thereof under stringent conditions.

[0015] Moreover, this invention relates to a method for determining achemotherapeutic regimen, comprising obtaining an mRNA sample from atumor specimen; determining DPD gene expression level in the specimen;comparing the determined DPD gene expression levels with a predeterminedthreshold level for that gene; and determining a chemotherapeuticregimen based on the results of the comparison of the determined geneexpression level with the predetermined threshold level.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a graph showing a comparison of four differentoligionucleotide primer pairs for their ability to amplify DPD mRNAderived from 10 different formalin-FPE tissue samples. Samples #1-5, and#8-10 are derived from colon tumor, #6 from bronchoalveolar tumor and #7from small bowel tumor biopsies. Oligonucleotide primer pairs DPD1(DPD-70F, (SEQ ID NO: 3) and DPD-201R, (SEQ ID NO: 4)), DPD2(DPD2p-1129F (SEQ ID NO: 5) and DPD2p-1208R (SEQ ID NO: 6)) are noteffective in measuring DPD mRNA levels in these samples. Oligonucleotideprimer pairs DPD3A (DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO:2)) and DPD3B (DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8))are effective in ascertaining DPD levels in various samples.

[0017]FIG. 2 is a graph showing a comparison of DPD mRNA amplificationefficiency of the oligonucleotide primer pairs DPD3A (DPD3a-51F (SEQ IDNO: 1) and DPD3a-134R (SEQ ID NO: 2)) and DPD1 (DPD-70F (SEQ ID NO: 3)and DPD-201R (SEQ ID NO: 4)) in frozen tissue samples. The graphillustrates that not only is the oligonucleotide primer pair DPD3A(DPD3a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2)) also effectivein measuring DPD expression levels in frozen tissue samples (as well asFPE derived samples) it is more efficient than the oligonucleotideprimer pair DPD1 (DPD-70F (SEQ ID NO: 3) and DPD-201R (SEQ ID NO: 4)).

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present inventors disclose oligonucleotide primers andoligonucleotide primers substantially identical thereto that allowaccurate assessment of DPD expression in tissues. These oligonucleotideprimers, DPD3 a-51F (SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2), (alsoreferred to herein as the oligonucleotide primer pair DPD3A) andoligionucleotide primers DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQID NO: 8), (also referred to herein as the oligonucleotide primer pairDPD3B) are particularly effective when used to measure DPD geneexpression in fixed paraffin embedded (FPE) tumor specimens.

[0019] “Substantially identical” in the nucleic acid context as usedherein, means that the oligonucleotides hybridizes to a target understringent conditions, and also that the nucleic acid segments, or theircomplementary strands, when compared, are the same when properlyaligned, with the appropriate nucleotide insertions and deletions, in atleast about 60% of the nucleotides, typically, at least about 70%, moretypically, at least about 80%, usually, at least about 90%, and moreusually, at least, about 95-98% of the nucleotides. Selectivehybridization exists when the hybridization is more selective than totallack of specificity. See, Kanehisa, Nucleic Acids Res., 12:203-213(1984).

[0020] This invention includes substantially identical oligonucleotidesthat hybridize under stringent conditions (as defined herein) to all ora portion of the oligonucleotide primer sequence of DPD3A-51F (SEQ IDNO:1), its complement, DPD3A-134R (SEQ ID NO: 2) or its complement.Furthermore, this invention also includes substantially identicaloligonucleotides that hybridize under stringent conditions (as definedherein) to all or portion of the oligonucleotide primer sequenceDPD3b-651F (SEQ ID NO: 7) its complement, DPD3b-736R (SEQ ID NO: 8), orits complement.

[0021] Under stringent hybridization conditions, only highlycomplementary, i.e., substantially similar nucleic acid sequenceshybridize. Preferably, such conditions prevent hybridization of nucleicacids having 4 or more mismatches out of 20 contiguous nucleotides, morepreferably 2 or more mismatches out of 20 contiguous nucleotides, mostpreferably one or more mismatch out of 20 contiguous nucleotides.

[0022] The hybridizing portion of the nucleic acids is typically atleast 10 (e.g., 15) nucleotides in length. The hybridizing portion ofthe hybridizing nucleic acid is at least about 80%, preferably at leastabout 95%, or most preferably about at least 98%, identical to thesequence of a portion or all of oligonucleotide primer DPD3A-51F (SEQ IDNO: 1), its complement, DPD3A-134R (SEQ ID NO: 2) or its complement.Additionally, the hybridizing portion of the hybridizing nucleic acid isat least least about 80%, preferably at least about 95%, or mostpreferably about at least 98%, identical to the sequence of a portion orall of oligonucleotide primer DPD3b-651F (SEQ ID NO: 7), its complement,DPD3b-736R (SEQ ID NO: 8) or its complement.

[0023] Hybridization of the oligonucleotide primer to a nucleic acidsample under stringent conditions is defined below. Nucleic acid duplexor hybrid stability is expressed as a melting temperature (T_(m)), whichis the temperture at which the probe dissociates from the target DNA.This melting temperature is used to define the required stringencyconditions. If sequences are to be identified that are substantiallyidentical to the probe, rather than identical, then it is useful tofirst establish the lowest temperature at which only holmologoushybridization occurs with a particular concentration of salt (e.g. SSCor SSPE). Then assuming that 1% mismatching results in a 1° C. decreasein T_(m), the temperatre of the final wash in the hybridization reactionis reduced accordingly (for example, if sequences having >95% identitywith the probe are sought, the final wash temperature is decrease by 5°C.). In practice, the change in Tm can be between 0.5° C. and 1.5° C.per 1% mismatch.

[0024] Stringent conditions involve hybridizing at 68° C. in 5× SSC/5×Denhart's solution/1.0% SDS, and washing in 0.2× SSC/0.1% SDS at roomtemperature. Moderately stringent conditions include washing in 3× SSCat 42° C. The parameters of salt concentration and temperature be variedto achieve optimal level of identity between the primer and the targetnucleic acid. Additional guidance regarding such conditions is readilyavailable in the art, for example, Sambrook, Fischer and Maniatis,Molecular Cloning, a laboratory manual, (2nd ed.), Cold Spring HarborLaboratory Press, New York, (1989) and F. M. Ausubel et al eds., CurrentProtocols in Molecular Biology, John Wiley and Sons (1994).

[0025] This aspect of the invention involves use of a method forreliable extraction of RNA from an FPE specimen and second,determination of the content of DPD mRNA in the specimen by usingoligonucleotide primers oligionucleotide primer pair DPD3A (DPD3a-51F(SEQ ID NO: 1) and DPD3a-134R (SEQ ID NO: 2)) or oligonucleotidessubstantially identical thereto or DPD3B (DPD3b-651F (SEQ ID NO: 7) andDPD3b-736R (SEQ ID NO: 8)) or oligonucleotides substantially identicalthereto, for carrying out reverse transcriptase polymerase chainreaction. RNA is extracted from the FPE cells by any of the methods formRNA isolation from such samples as described in U.S. patent applicationSer. No. 09/469,338, filed Dec. 20, 1999, and is hereby incorporated byreference in its entirety.

[0026] The present invention resides in part in the finding that therelative amount of DPD mRNA is correlated with resistance to thechemotherapeutic agent 5-FU. It has been found herein that tumorsexpressing high levels of DPD mRNA are considered likely to be resistantto 5-FU. Conversely, those tumors expressing low amounts of DPD mRNA arelikely to be sensitive to 5-FU. A patient's relative expression of tumorDPD mRNA is judged by comparing it to a predetermined thresholdexpression level of expression of DPD.

[0027] A predetermined threshold level of DPD mRNA expression, asdefined herein, is a level of DPD expression above which it has beenfound that tumors are likely to be resistant to 5-FU. Expression levelsbelow this threshold level are likely to be found in tumors sensitive to5-FU. The range of relative expression of DPD, expressed as a ratio ofDPD: an internal control gene, among tumors responding to a 5-FU basedchemotherapeutic regimen responding tumors of less than about 0.6 toabout 2.5, (about a 4.2-fold range). Tumors not responding to a 5-FUbased chemotherapeutic regimen have relative expression of DPD : aninternal control gene ratio of about 0.2 to about 16 (about an 80-foldrange). Tumors generally do not respond to 5-FU treatment if there isDPD expression greater than about 2.0, preferably greater than about2.5.

[0028] “Substantially equivalent” as used herein refers to a thresholdlevel of DPD expression level that is about 2.0 to about 2.5.

[0029] The oligonucleotide primers of the invention enable that allowaccurate assessment of DPD expression in a fixed paraffin embedded (FPE)tissue. FIG. 1. Additionally, the oligonucleotide primers of the presentinvention have been shown to be accurate for determining DPD expressionlevels in fresh or frozen tissue, i.e. they have high specificity fortheir target RNA. Thus, methods of the invention are not limited to useof paraffin embedded tissue. Oligonucleotide primers disclosed hereinare capable of allowing accurate assessment of DPD gene expression in afixed paraffin embedded tissue, as well as in frozen or fresh tissue.FIG. 2. This is due to the fact that the mRNA derived from FPE samplesis more fragmented relative to that of fresh or frozen tissue and istherefore, more difficult to quantify. Thus, the present inventionprovides oligonucleotide primers that are suitable for use in assayingDPD expression levels in FPE tissue, where previously there existed nosuitable assay. See FIG. 1.

[0030] Expression of DPD mRNA is correlated with clinical resistance to5-FU-based chemotherapy. In particular, expression of high levels of DPDmRNA correlate with resistance to 5-FU-based chemotherapies. The presentmethods can be applied to any type of tissue. For example, forexamination of resistance of tumor tissue, it is desirable to examinethe tumor tissue. Preferably, it is desirable to also examine a portionof normal tissue from the patient from which the tumor is obtained.Patients whose normal tissues are resistant to 5-FU-basedchemotherapeutic compounds, but whose tumors are expected to besensitive to such compounds, may then be treated with higher amounts ofthe chemotherapeutic composition.

[0031] The methods in this invention are applied over a wide range oftumor types. This allows for the preparation of individual “tumorexpression profiles” whereby expression levels of DPD may be determinedin individual patient samples and response to various chemotherapeuticscan be predicted. Most preferably, the methods of the present inventionare applied to bronchalveolar, small bowel or colon tumors. Forapplication of some embodiments of the invention to particular tumortypes, it is preferable to confirm the relationship of the measurementto clinical resistance by compiling a data-set of the correlation of theparticular DPD expression parameter measured and clinical resistance to5-FU-based chemotherapy.

[0032] The methods of the present invention include the step ofobtaining sample of cells from a patient's tumor. Solid or lymphoidtumors, or parts thereof are surgically resected from the patient. If itis not possible to extract RNA from the tissue sample soon after itsresection, the sample may then be fixed or frozen. It will then be usedto obtain RNA. RNA extracted and isolated from frozen or fresh samplesof resected tissue is extracted by any of the methods typical in theart, for example, Sambrook, Fischer and Maniatis, Molecular Cloning, alaboratory manual, (2nd ed.), Cold Spring Harbor Laboratory Press, NewYork, (1989). Preferably, care is taken to avoid degradation of RNAduring the extraction process.

[0033] Tissue obtained from the patient may fixed, preferably byformalin (formaldehyde) or gluteraldehyde treatment, for example.Biological samples fixed by alcohol immersion are also contemplated inthe present invention. Fixed biological samples are often dehydrated andembedded in paraffin or other solid supports known to those of skill inthe art. Such solid supports are envisioned to be removable with organicsolvents, allowing for subsequent rehydration of preserved tissue. Fixedand paraffin-embedded (FPE) tissue specimen as described herein refersto storable or archival tissue samples.

[0034] RNA is extracted from the FPE cells by any of the methods asdescribed in U.S. patent application Ser. No. 09/469,338, filed Dec. 20,1999, which is hereby incorporated by reference in its entirety. Mostpreferably, RNA is extracted from tumor cells from a formalin-fixed andparaffin-embedded tissue specimen.

[0035] In an embodiment of the invention, RNA is isolated from anarchival pathological sample or biopsy which is first deparaffinized. Anexemplary deparaffinization method involves washing the paraffinizedsample with an organic solvent, such as xylene. Deparaffinized samplescan be rehydrated with an aqueous solution of a lower alcohol. Suitablelower alcohols, for example include, methanol, ethanol, propanols, andbutanols. Deparaffinized samples may be rehydrated with successivewashes with lower alcoholic solutions of decreasing concentration.Alternatively, the sample is simultaneously deparaffinized andrehydrated.

[0036] Once the sample is reyhdrated, RNA is extracted from therehydrated tissue. Deparaffinized samples can be homogenized usingmechanical, sonic or other means of homogenization. In one embodiment,rehydrated samples are homogenized in a solution comprising a chaotropicagent, such as guanidinium thiocyanate (also sold as guanidiniumisothiocyanate).

[0037] An “effective concentration of chaotropic agent” is chosen suchthat at an RNA is purified from a paraffin-embedded sample in an amountof greater than about 10 fold that isolated in the absence of achaotropic agent. Chaotropic agents include: guanidinium compounds,urea, formamide, potassium iodiode, potassium thiocyantate and similarcompounds. The preferred chaotropic agent for the methods of theinvention is a guanidinium compound, such as guanidinium isothiocyanate(also sold as guanidinium thiocyanate) and guanidinium hydrochloride.Many anionic counterions are useful, and one of skill in the art canprepare many guanidinium salts with such appropriate anions. Theeffective concentration of guanidinium solution used in the inventiongenerally has a concentration in the range of about 1 to about 5M with apreferred value of about 4M. If RNA is already in solution, theguanidinium solution may be of higher concentration such that the finalconcentration achieved in the sample is in the range of about 1 to about5M. The guanidinium solution also is preferably buffered to a pH ofabout 3 to about 6, more preferably about 4, with a suitable biochemicalbuffer such as Tris-Cl. The chaotropic solution may also containreducing agents, such as dithiothreitol (DTT) and (β-mercaptoethanol(BME). The chaotropic solution may also contain RNAse inhibitors.

[0038] Homogenized samples may be heated to a temperature in the rangeof about 50 to about 100° C. in a chaotropic solution, which contains aneffective amount of a chaotropic agent, such as a guanidinium compound.A preferred chaotropic agent is guanidinium thiocyanate.

[0039] RNA is then recovered from the solution by, for example, phenolchloroform extraction, ion exchange chromatography or size-exclusionchromatography. RNA may then be further purified using the techniques ofextraction, electrophoresis, chromatography, precipitation or othersuitable techniques.

[0040] The quantification of DPD mRNA from purified total mRNA fromfresh, frozen or fixed is preferably carried out usingreverse-transcriptase polymerase chain reaction (RT-PCR) methods commonin the art, for example. Other methods of quantifying of DPD mRNAinclude for example, the use of molecular beacons and other labeledprobes useful in multiplex PCR. Additionally, the present inventionenvisages the quantification of DPD mRNA via use of a PCR-free systemsemploying, for example fluorescent labeled probes similar to those ofthe Invader® Assay (Third Wave Technologies, Inc.). Most preferably,quantification of DPD cDNA and an internal control or house keeping gene(e.g. β-actin) is done using a fluorescence based real-time detectionmethod (ABI PRISM 7700 or 7900 Sequence Detection System [TaqMan®],Applied Biosystems, Foster City, Calif.) or similar system as describedby Heid et al., (Genome Res 1996;6:986-994) and Gibson et al.(Genome Res1996;6:995-1001).

[0041] As used herein, a “house keeping” gene or “internal control” ismeant to include any constitutively or globally expressed gene whosepresence enables normalization of mRNA levels. Normalization is adetermination of the overall constitutive level of gene transcriptionand a control for variations in RNA recovery. “House-keeping” genes or“internal controls” can include, but are not limited to the cyclophilingene, β-actin gene, the transferrin receptor gene, GAPDH gene, and thelike. Most preferably, the internal control gene is β-actin gene asdescribed by Eads et al., Cancer Research 1999; 59:2302-2306.

[0042] The methods of the invention are applicable to a wide range oftissue and tumor types and so can be used for assessment of treatment ina patient and as a diagnostic or prognostic tool in a range of cancersincluding breast, head and neck, lung, esophageal, colorectal, andothers. Preferably, the present methods are applied to prognosis ofbronchoalveolar, small bowel, and colon cancer.

[0043] From the measurement of the amount of DPD mRNA that is expressedin the tumor, the skilled practitioner one can make a prognosisconcerning clinical resistance of a tumor to 5-FU-based chemotherapy.5-FU-based chemotherapy comprises administration of 5-FU, itsderivatives, alone or with other chemotherapeutics or with a DPDinhibitor such as uracil, 5-ethynyluracil, bromovinyluracil, thymine,benzyloxybenzyluracil (BBU) or 5-chloro-2,4-dihydroxypyridine.Furthermore, it has been found that co-administration of a5′-deoxy-cytidine derivative of the formula (I) with 5-FU or aderivative thereof results in significantly improved delivery of 5-FUselectively to tumor tissues as compared with the combination of 5-FU orits derivative with a DPD inhibitor 5-ethynyluracil, and showssignificantly improved antitumor activity in human cancer xenograftmodels.

[0044] The invention being thus described, practice of the invention isillustrated by the experimental examples provided below. The skilledpractitioner will realize that the materials and methods used in theillustrative examples can be modified in various ways. Suchmodifications are considered to fall within the scope of the presentinvention.

EXAMPLES Example 1 RNA Isolation from FPE Tissue

[0045] RNA is extracted from paraffin-embedded tissue by the followinggeneral procedure.

[0046] A. Deparaffinization and Hydration of Sections:

[0047] (1) A portion of an approximately 10 μM section is placed in a1.5 mL plastic centrifuge tube.

[0048] (2) 600 μL, of xylene are added and the mixture is shakenvigorously for about 10 minutes at room temperature (roughly 20 to 25°C.).

[0049] (3) The sample is centrifuged for about 7 minutes at roomtemperature at the maximum speed of the bench top centrifuge (about10-20,000× g).

[0050] (4) Steps 2 and 3 are repeated until the majority of paraffin hasbeen dissolved. Two or more times are normally required depending on theamount of paraffin included in the original sample portion.

[0051] (5) The xylene solution is removed by vigorously shaking with alower alcohol, preferably with 100% ethanol (about 600 μL) for about 3minutes.

[0052] (6) The tube is centrifuged for about 7 minutes as in step (3).The supernatant is decanted and discarded. The pellet becomes white.

[0053] (7) Steps 5 and 6 are repeated with successively more diluteethanol solutions: first with about 95% ethanol, then with about 80% andfinally with about 70% ethanol.

[0054] (8) The sample is centrifuged for 7 minutes at room temperatureas in step (3). The supernatant is discarded and the pellet is allowedto dry at room temperature for about 5 minutes.

[0055] B. RNA Isolation with Phenol-Chloroform

[0056] (1) 400 μL guanidine isothiocyanate solution including 0.5%sarcosine and 8 μL dithiothreitol is added.

[0057] (2) The sample is then homogenized with a tissue homogenizer(Ultra-Turrax, IKA-Works, Inc., Wilmington, N.C.) for about 2 to 3minutes while gradually increasing the speed from low speed (speed 1) tohigh speed (speed 5).

[0058] (3) The sample is then heated at about 95° C. for about 5-20minutes. It is preferable to pierce the cap of the tube containing thesample before heating with a fine gauge needle. Alternatively, the capmay be affixed with a plastic clamp or with laboratory film.

[0059] (4) The sample is then extracted with 50 μL 2M sodium acetate atpH 4.0 and 600 μL of phenol/chloroform/isoamyl alcohol (10:1.93:0.036),prepared fresh by mixing 18 mL phenol with 3.6 mL of a 1:49 isoamylalcohol:chloroform solution. The solution is shaken vigorously for about10 seconds then cooled on ice for about 15 minutes.

[0060] (5) The solution is centrifuged for about 7 minutes at maximumspeed. The upper (aqueous) phase is transferred to a new tube.

[0061] (6) The RNA is precipitated with about 10 μL glycogen and with400 μL isopropanol for 30 minutes at -20° C.

[0062] (7) The RNA is pelleted by centrifugation for about 7 minutes ina benchtop centrifuge at maximum speed; the supernatant is decanted anddiscarded; and the pellet washed with approximately 500 μL of about 70to 75% ethanol.

[0063] (8) The sample is centrifuged again for 7 minutes at maximumspeed. The supernatant is decanted and the pellet air dried. The pelletis then dissolved in an appropriate buffer for further experiments (e.g.50 pI. 5 mM Tris chloride, pH 8.0).

Example 2 mRNA Reverse Transcription and PCR

[0064] Reverse Transcription:

[0065] RNA was isolated from microdissected or non-microdissectedformalin fixed paraffin embedded (FPE) tissue as illustrated in Example1 and as previously described in U.S. application Ser. No. 09/469,338filed Dec. 20, 1999, which is hereby incorporated by reference in itsentirety. After precipitation with ethanol and centrifugation, the RNApellet was dissolved in 50 ul of 5 mM Tris/Cl at pH 8.0. The resultingRNA was reverse transcribed with random hexamers and M-MLV from LifeTechnologies (CAT#28025-02.). The reverse transcription was accomplishedby mixing 25 μl of the RNA solution with 25.5 μl of “reversetranscription mix” (see below). The reaction was placed in athermocycler for 8 min at 26° C. (for binding the random hexamers toRNA), 45 min at 42° C. (for the M-MLV reverse transcription enzymaticreaction) and 5 min at 95° C. (for heat inactivation of DNAse).

[0066] “Reverse transcription mix” consisted of 10 ul 5× buffer (250 mMTris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2), 0.5 ul random hexamers (50O.D. dissolved in 550 ul of 10 mM Tris-HCl pH 7.5) 5 ul 10 mM dNTPs(dATP, dGTP, dCTP and dTTP), 5 ul 0.1 M DTT, 1.25 ul BSA (3 mg/ml in 10mM Tris-HCL, pH 7.5), 1.25 ul RNA Guard 24,800U/ml (RNAse inhibitor)(Porcine #27-0816, Amersham Pharmacia) and 2.5 ul MMLV 200U/ul (LifeTech Cat #28025-02).

[0067] Final concentrations of reaction components were: 50 mM Tris-HCl,pH 8.3, 75 mM KCl, 3 mM MgCl2, 1.0 mM dNTP, 1.0 mM DTT, 0.00375. mg/mlBSA, 0.62 U/ul RNA Guard and 10 U/ul MMLV.

[0068] PCR Quantification of mRNA Expression:

[0069] Quantification of DPD cDNA and an internal control or housekeeping gene (i.e. β-actin, as described in Eads et al., (CancerResearch 1999; 59:2302-2306) was done using a fluorescence basedreal-time detection method (ABI PRISM 7700 or 7900 Sequence DetectionSystem [TaqMan®], Applied Biosystems, Foster City, Calif.) as describedby Heid et al., (Genome Res 1996;6:986-994); Gibson et al., (Genome Res1996;6:995-1001). In brief, this method uses a dual labelled fluorogenicoligonucleotide probe (the TaqMan® probe) that anneals specificallywithin the template amplicon spanning the forward and reverse primers.Laser stimulation within the capped wells containing the reactionmixture causes emission of a 3′ quencher dye (TAMRA) until the probe iscleaved by the 5′ to 3′ nuclease activity of the DNA polymerase duringPCR extension, causing release of a 5′ reporter dye (6FAM). Productionof an amplicon thus causes emission of a fluorescent signal that isdetected by the TaqMan®'s CCD (charge-coupled device) detection camera,and the amount of signal produced at a threshold cycle within the purelyexponential phase of the PCR reaction reflecting the starting copynumber of the sequence of interest. TaqMan® probe for theoligonucleotide primer pair DPD1 (DPD-70F (SEQ ID NO: 3) and DPD-201R(SEQ ID NO: 4)) is DPD-108Tc (SEQ ID NO:9). TaqMan® probe for theoligonucleotide primer pair DPD2 (DPD2p-1129F (SEQ ID NO: 5) andDPD2p-1208R (SEQ ID NO: 6)) is DPD-2p-1154Tc (SEQ ID NO: 10). TaqMan®probe for the oligonucleotide primer pair DPD3A (DPD3a-51F (SEQ IDNO: 1) and DPD3a-134R (SEQ ID NO: 2)) is DPD3A-71Tc (SEQ ID NO: 11).TaqMan® probe for the oligonucleotide primer pair DPD3B (DPD3b-651F (SEQID NO: 7) and DPD3b-736R (SEQ ID NO: 8)) is DPD3b-685Tc (SEQ ID NO: 12).

[0070] Comparison of the starting copy number of the sequence ofinterest with the starting copy number of the internal control geneprovided a relative gene expression level. TaqMan® analyses yield valuesthat are expressed as ratios between two absolute measurements (gene ofinterest/internal control gene).

[0071] The PCR reaction contained olgionulceotide primers from the pairDPD1 (DPD-70F (SEQ ID NO: 3) and DPD-201R (SEQ ID NO: 4)); DPD2(DPD2p-1129F (SEQ ID NO: 5) and DPD2p-1208R (SEQ ID NO: 6)); DPD3B(DPD3b-651F (SEQ ID NO: 7), T_(m)=58° C. and DPD3b-736R (SEQ ID NO: 8),T_(m)=60° C.); or oligonucleotide primer pair DPD3A (DPD3a-51F (SEQ IDNO: 1), T_(m)=59° C. and DPD3a-134R (SEQ ID NO: 2), T_(m)=59° C.). EachPCR reaction mixture consisted 0.5 μl of the reverse transcriptionreaction containing the cDNA as well as 600 nM each of botholigonucleotide primers from only one pair (DPD1, DPD2, DPD3B or DPD3A),200 nM corresponding TaqMan® probe (for either DPD1, DPD2, DPD3B orDPD3A), 5 U AmpliTaq Gold Polymerase, 200 μM each dATP, dCTP, dGTP, 400μM dTTP, 5.5 mM MgCl₂, and 1× Taqman Buffer A containing a referencedye, to a final volume of less than or equal to 25 μl (all reagents,Applied Biosystems, Foster City, Calif.). Cycling conditions were, 95°C. for 10 min, followed by 45 cycles at 95° C. for 15s and 60° C. for 1min.

Example 3 DPD Expression in FPE Tumor Samples

[0072] The oligonucleotide primer pairs DPD3A (DPD3a-51F (SEQ ID NO: 1)and DPD3a-13R (SEQ ID NO: 2)) and DPD3B (DPD3b-651F (SEQ ID NO: 7) andDPD3b-736R (SEQ ID NO: 8)) allowed robust, reproducible quantitation ofDPD gene expression by RT-PCR using RNA extracted from paraffin-embeddedtissue. FIG. 1. Oligonucleotide primer pair DPD3A (DPD3 a-51F (SEQ IDNO: 1) and DPD3a-13R (SEQ ID NO: 2)) also significantly increased thesensitivity of DPD gene expression analysis by RT-PCR in fresh frozentissue. FIG. 2. RT-PCR was performed using the ABI Prism 7700 SequenceDetection System (Taqman®) as described in Example 2, above.

[0073] Thirty cycles were used in the PCR reaction. Each cycle consistedof denaturing at 96° C. for 1 min, annealing at 55° C. for 1 min andextending at 72° C. for 2 min. The amplified product usingoligionucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1) andDPD3a-13R (SEQ ID NO: 2)) was 84 base pairs in length. The amplifiedproduct corresponded to region of DPD cDNA spanning a portion of the 5′untranslated region (UTR) and running into Exon 1. The amplified productusing oligionucleotide primer pair DPD3B (DPD3b-651F (SEQ ID NO: 7) andDPD3b-736R (SEQ ID NO: 8)) is 86 base pairs in length. The amplifiedproduct corresponded to amplifies a region of DPD cDNA corresponding toExon 6.

[0074] Oligonucleotide primer pairs DPD3A (DPD3a-51 F (SEQ ID NO: 1) andDPD3a-13R (SEQ ID NO: 2)) and DPD3B (DPD3b-651F (SEQ ID NO: 7), andDPD3b-736R (SEQ ID NO: 8)) were compared to other existing primer setsfor their ability to amplify DPD mRNA derived from 10 different FPEtissue samples. Samples #1-5, and #8-10 were derived from colon, #6 frombronchoalveolar and #7 from small bowel tumor biopsies. Otheroligonucleic acid primer pairs used were DPD1 (DPD-70F (SEQ ID NO: 3)and DPD-201R (SEQ ID NO: 4)) and DPD2 (DPD2p-1129F (SEQ ID NO: 5) andDPD2p-1208R (SEQ ID NO: 6)).

[0075] The oligonucleotide primer pair DPD3A (DPD3a-51F (SEQ ID NO: 1)and DPD3a-134R (SEQ ID NO: 2)) was most effective in accuratelyascertaining DPD levels in various samples. Oligionucleotide primer pairDPD3B (DPD3b-651F (SEQ ID NO: 7) and DPD3b-736R (SEQ ID NO: 8)) was alsoeffective, yet did not provide as strong a signal. Results illustratedin FIG. 1.

1 12 1 19 DNA Artificial Sequence Oligonucleotide Primer 1 aggacgcaaggagggtttg 19 2 20 DNA Artificial Sequence Oligonucleotide Primer 2gtccgccgag tccttactga 20 3 22 DNA Artificial Sequence OligonucleotidePrimer 3 tcactggcag actcgagact gt 22 4 18 DNA Artificial SequenceOligonucleotide Primer 4 tggccgaagt ggaacaca 18 5 22 DNA ArtificialSequence Oligonucleotide Primer 5 ctgcctttga ctgtgcaaca tc 22 6 27 DNAArtificial Sequence Oligonucleotide Primer 6 attaacaaag ccttttctgaagacgat 27 7 23 DNA Artificial Sequence Oligonucleotide Primer 7gaagcctatt ctgcaaagat tgc 23 8 21 DNA Artificial SequenceOligonucleotide Primer 8 gagtacccca atcgagccaa a 21 9 25 DNA ArtificialSequence Oligonucleotide Primer 9 ccgccgagtc cttactgagc acagg 25 10 25DNA Artificial Sequence Oligonucleotide Primer 10 cacacggcga gctccacaacgtaga 25 11 29 DNA Artificial Sequence Oligonucleotide Primer 11cagtgcctac agtctcgagt ctgccagtg 29 12 31 DNA Artificial SequenceOligonucleotide Primer 12 aaggaagcac aacttatact tgcaggccca g 31

1. An oligonucleotide having the sequence of SEQ ID NO:1 or which issubstantially identical thereto.
 2. An oligonucleotide having thesequence of SEQ ID NO:2 or which is substantially identical thereto. 3.An oligonucleotide having the sequence of SEQ ID NO:7 or which issubstantially identically thereto.
 4. An oligonucleotide having thesequence of SEQ ID NO:8 or which is substantially identically thereto.5. A kit for detecting expression of a Dihydropyrimidine dehydrogenase(DPD) gene in a tissue obtained from a patient comprising theoligonucleotide pair DPD3A or a pair of oligonucleotides substantiallyidentical thereto or the oligonucleotide pair DPD3B or a pair ofoligonucleotides substantially identical thereto.
 6. A method ofdetermining the relative level of Dihydropyrimidine dehydrogenase (DPD)gene expression in a tissue sample comprising: (a) obtaining a tumorsample from a patient; (b) isolating mRNA from said tumor sample; (c)amplifying the mRNA using an oligonucleotide primer having the sequenceof SEQ ID: 1, or which is substantially identical thereto and anoligonucleotide having the sequence SEQ ID: 2, or which is substantiallyidentical thereto; (d) comparing the amount of Dihydropyrimidinedehydrogenase (DPD) mRNA from step (c) to an amount of mRNA of aninternal control gene.
 7. The method of claim 6, wherein the tumorsample is frozen after being obtained from the patient.
 8. The method ofclaim 6, wherein the tumor sample is fixed after being obtained from thepatient.
 9. The method of claim 8, wherein the tumor sample is embeddedin paraffin fixed after being fixed.
 10. The method of claim 8 or 9,wherein the RNA is isolated in the presence of an effective amount ofchaotropic agent.
 11. The method of any one of claims 6, 8, or 9,wherein a tumor sample comprises non-tumor tissue and tumor tissue. 12.A method for determining a 5-Fluorouracil-based chemotherapeutic regimenfor treating a tumor in patient comprising: (a) obtaining a tumor samplefrom the tumor; (b) isolating mRNA from said tumor sample; (c)subjecting the mRNA to amplification using a pair of oligonucleotideprimers having of the sequence of SEQ ID: 1, or which is substantiallyidentical thereto and an oligonucleotide having the sequence SEQ ID: 2,or which is substantially identical thereto to obtain an amplifiedsample, (d) determining the amount of Dihydropyrimidine dehydrogenase(DPD) mRNA in the amplified sample; (e) comparing the amount ofDihydropyrimidine dehydrogenase (DPD) mRNA in the amplified sample witha predetermined threshold level for DPD expression; (f) determining a5-Fluorouracil-based chemotherapeutic regimen for said patient based onthe difference in amount of DPD mRNA in the amplified sample and thethreshold level for DPD gene expression.
 13. The method of claim 12,wherein said predetermined threshold level of DPD gene expression isabout 2.0 to about 2.5 times internal control gene expression level. 14.The method of claim 12 or 13, wherein said internal control gene isβ-actin.
 15. The method of claim 13, wherein the tumor sample is fixedand embedded after being obtained.
 16. The method of claim 13, whereinthe mRNA is isolated in the presence of an effective amount ofchaotropic agent.
 17. A method of determining the relative level ofDihydropyrimidine dehydrogenase (DPD) gene expression in a tissue samplecomprising; (a) obtaining a tumor sample from a patient; (b) isolatingmRNA from said tumor sample; (c) amplifying the mRNA using anoligonucleotide primer having the sequence of SEQ ID: 7, or which issubstantially identical thereto and an oligonucleotide having thesequence SEQ ID: 8, or which is substantially identical thereto; (d)comparing the amount of the mRNA from step (c) to an amount of mRNA ofan internal control.
 18. The method of claim 17, wherein the tumorsample is frozen after being obtained from the patient.
 19. The methodof claim 17, wherein the a tumor sample is embedded in paraffin fixedafter being fixed.
 20. The method of claim 19, wherein the mRNA isisolated in the presence of an effective amount of chaotropic agent. 21.The method of claim 17 wherein the tissue sample is obtained from atumor.
 22. The method of claim 20, wherein a tumor sample comprisesnon-tumor tissue and tumor tissue.
 23. A method for determining a5-Fluorouracil-based chemotherapeutic regimen for treating a tumor in apatient comprising: (a) obtaining a tumor sample from the tumor; (b)isolating mRNA from a tumor sample; (c) subjecting the mRNA toamplification using a pair of oligonucleotide primers having of thesequence of SEQ ID: 7, or which is substantially identical thereto andan oligonucleotide having the sequence SEQ ID: 8, or which issubstantially identical thereto, to obtain an amplified sample; (d)determining the amount of Dihydropyrimidine dehydrogenase (DPD) mRNA inthe amplified sample; (e) comparing the amount of Dihydropyrimidinedehydrogenase (DPD) mRNA in the amplified sample with a predeterminedthreshold level for DPD expression; (f) determining a5-Fluorouracil-based chemotherapeutic regimen for said patient based onthe difference in amount of DPD mRNA in the amplified sample and thethreshold level for DPD gene expression.
 24. The method of claim 23,wherein said predetermined threshold level of DPD gene expression isabout 2.0 to about 2.5 times internal control gene expression level. 25.The method of claim 23, or 24, wherein said internal control gene isβ-actin.
 26. The method of any one of claims 5, 6, 12, 17, or 23;wherein the at least one tissue sample contains bronchoalveolar tumortissue, small bowel tumor tissue or colon tumor tissue.